Synthesis and characterization of polysaccharide- and protein-based edible films and application as packaging materials for fresh fish fillets

The rising packaging industry together with global demand for sustainable production has increased the interest in developing biodegradable packaging materials. The aim of the study was to develop edible films based on pectin, gelatin, and hydroxypropyl methylcellulose and evaluate their applicability as biodegradable packaging materials for gilthead seabream fillets. Mechanical properties, water barriers, wettability of the films through contact angle measurement, optical, and UV–Vis barrier properties were evaluated for food packaging applications. The effective blend of polysaccharide and protein film-forming solutions was confirmed by the produced films with excellent optical properties, acceptable mechanical properties and adequate barriers to water vapor. The contact angle for pectin based and gelatin based films were higher than 90° indicating the hydrophobic films, while HPMC based films had contact angle lower than 90°. The produced films were tested as alternative and environmentally friendly packaging materials for gilthead seabream fillets during refrigerated storage. All tested packaging conditions resulted in similar shelf-life in packed gilthead seabream fillets (i.e. 7–8 days at 2 °C). The results showed that the developed films may reduce the use of conventional petroleum-based food packaging materials without affecting the shelf-life of fish.

www.nature.com/scientificreports/Cellulose is a polysaccharide commonly used in food applications, which has been reported as alternative food packaging material for the replacement of conventional petrochemical based plastics 5 .HPMC can be produced by cellulose modification by an alkaline treatment.HPMC can be produced using a 18% sodium hydroxide solution in purified wood pulps.Methyl and hydroxypropyl ether groups are introduced into cellulose, due to the reaction of alkali cellulose with propylene oxide followed with reaction with methyl chloride.HPMC is an odorless and tasteless compound, and thus forms films with neutral organoleptic characteristics, considering odor and taste 6 .
Pectin is a cell wall polysaccharide, which is essential for primary cell wall extension and plant growth.Pectin may be extracted from citrus peel, exhibits high solubility in water and is insoluble in organic systems.Pectin mainly consists of galacturonic acid (70%), forming linear anionic backbone chains without side parts and nonanionic regions.The free carboxyl groups in the pectin structure provide a solution with acidic pH.Pectin has been reported as an appropriate compound for packaging film formation, mainly due to its compatibility with other natural polysaccharides, proteins or lipids 7 .
Proteins are used in packaging field as they have the ability to form biodegradable films.Films based on proteins have satisfactory mechanical and adequate gas barrier properties 8 .
Gelatin is reported as an alternative biopolymer, appropriate for food packaging applications, with the aim to enhance the functionality of packed products.It is considered as a water-soluble protein, obtained from partial hydrolysis of collagen.Because of its hydroscopic nature, gelatin-based films tend to dissolve in environments with increased moisture levels.The overall properties of gelatin-based films are dependent on molecular weight distribution and amino acid synthesis, directly related to the gelatin source, the extraction method and processing conditions 9 .Gelatin as raw material has no antimicrobial properties, however, antioxidant and antimicrobial compounds such as citrus essential oils and carvacrol may be incorporated into gelatin-based film forming solutions 8 .
Polysaccharides and protein blend materials have been tested for their biocompatibility to form packaging films.Generally, in composite films, each biopolymer gives its own unique characteristics, so the properties of the films are improved compared to single-material systems, considering the compatibility between the used biopolymers.There are three possible types of biopolymers that can improve the properties of single edible films: protein-protein, polysaccharide-polysaccharide and protein-polysaccharide 10 .Gelatin has low thermal and weak mechanical properties, while HPMC has gelation properties.In order to overcome these disadvantages HPMC and gelatin can be blended to form a continuous matrix 11 .Gelatin and pectin could produce value-added packaging materials since having excellent film forming properties and produce compatible films, as reported in the literature 12,13 .
Fish and seafood products are important commodities due to their high nutritional and commercial value.The high water activity values (0.95-0.98) of fish and seafood together with the increased concentrations of nutrients result in the perishability of these products and their limited shelf-life.Globally, 35% of the total fish production is wasted before consumption 14 .For this reason, it is crucial to develop packaging materials that are environmentally friendly and can adequately maintain the freshness of fish products 15 .
The fabrication of HPMC, pectin, and gelatin by solvent casting method as separate films has been studied but their compatibility and characterization as composite films has not been yet justified in the literature 9,10,12,16 .Gelatin, pectin, and HPMC are three biopolymers which are dissolved in water and their films are characterized as hydrophilic and with high water content 17,18 .Although high water content is linked with biodegradability of the films, this may result in limitations in their application as food packaging materials, especially for foods with high moisture content.By mixing different proteins and polysaccharides, the chains of the polymers matrix are reorganized to the air-side interface, in order to form a more hydrophobic surface 19,20 .Another potential difficulty is the testing of the materials in sensitive food products, their applicability as alternative packaging systems has not been yet reported for fish products 10 .
The aim of the study was to develop and characterize composite films based on pectin, gelatin, and HPMC.The films were evaluated as packaging materials based on their mechanical properties, optical properties-and UV-Vis transmittance, water barriers, and hydrophilicity.Three different film formulations (HPMC-gelatin, HPMC-pectin, pectin-gelatin) were evaluated for their applicability as alternative packaging materials for chilled gilthead seabream (Sparus aurata) fillets during refrigerated storage at 2 °C as compared to a conventional polyvinyl chloride (PVC) film.

FT-IR analysis
The FT-IR spectra are presented in Fig. 1a,b.A main difference exists between HPMC, glycerol and HPMCglycerol spectra and was found at 3314-3377 cm −1 and it has been associated with the O-H stretching 21,22 .This difference may be related to the intermolecular hydrogen bonds between the -OH of HPMC and glycerol.
In the same spectral region and for the same reason, differentiation occurred (3341-3280 cm −1 ) between the pectin and the films were made of HPCM-glycerol, and the HPMC-pectin-glycerol FT-IR spectra.Moreover, for the same substances, differentiation also appeared in the peak 1017-1038 cm −1 (C-O stretching) [21][22][23] .According to the literature, pure HPMC films showed a strong absorption band at 1049 cm −1 and 945 cm −1 linked with ν as (C-O-C) of pyranose ring skeletal vibrations and ether linkages, respectively.Peaks at both wavelengths indicate the presence of ether groups in the HPMC structure 19 .
In spectra of HPMC and HPMC-glycerol a peak appeared at 1650 cm −1 which is due to the bending vibration of absorbed water 25 .In gelatin spectrum, the peaks were at 1697 (amide I) and 1535 cm −1 (amide II) 26 .These peaks shifted to 1643 and 1548 cm −1 in the films based on HPMC-glycerol-gelatin, probably due to the hydrogen bonds that developed.Wang et al. investigated the synthesis of HPMC films grafted by gelatin and they used glycerol as a plasticizer.The FT-IR analysis showed that the hydroxyl groups that are present in the HPMC and gelatin molecules had peak at 3280 cm −1 .Pure HPMC films had peaks at 3460 and 1040 cm −1 due to the stretching vibration of the hydroxyl groups and ether bond groups, respectively.When the HPMC films grafted with gelatin the peaks moved at 1547 cm −1 and 1240 cm −1 .These two peaks are the characteristic peaks of amide II band (relating to the bending vibration of N-H) and amide III band (relating to stretching vibration of C-N), and they are attributed to peptide bond of gelatin.The authors also pointed that by adding gelatin in HPMC matrix the intensity of peaks near 1040 cm −1 was lower while the intensity of peaks near 1645 cm −1 was higher 27 .This result is also observed in the present study as it is shown in Fig. 1a,b.
The FT-IR spectra pectin, glycerol and pectin-glycerol differentiated in spectral region 3314-3280 cm −1 and at 1017-1035 cm −1 .The gelatin spectrum appeared a peak at 3456 cm −1 which corresponds to N-H stretching.The pectin-glycerol spectrum showed a peak at 3280 cm −1 (O-H stretching).We observed that the pectinglycerol-gelatin spectrum presents an absorption at 3314 cm −1 .This shift correlated with hydrogen bonding.Another differentiation between pectin-glycerol and pectin-glycerol-gelatin spectra presented in the spectral region 1018-1025 cm −1 (C-O stretching).Ezati et al. studied the chemical interactions between pectin and gelatin using FT-IR analysis.They pointed peaks at 3248, 2936 and 2880 cm −1 due to O-H stretching and the intermolecular or intramolecular H-bonding, and C-H stretching vibration of methylene and methyl groups that were appeared in pectin.Peak at 1454 cm −1 was also due to pectin's carboxyl groups.Peaks at 1634, 1548 and 1236 cm −1 were due to the amide I (C=O stretching), amide II (N-H stretching) and amide III (C-N and N-H stretching) of gelatin 12 .
From the above discussion it is concluded that the complexes HPMC-glycerol, HPMC-gelatin-glycerol, pectin-gelatin, pectin-glycerol-gelatin and gelatin-glycerol have been formed in the developed packaging materials.

Thickness and mechanical properties
The films used as packaging materials should have adequate mechanical properties to protect the food from mechanical stresses during storage and handling.Elastic modulus (MPa), tensile strength (MPa), and elongation at break (%) can describe the integrity of the films under certain circumstances 28 .The mechanical properties and the thickness of the films are reported in Table 1.The thickness of the films in this study ranged between 31.72 μm and 35.31 μm.The mechanical properties were significantly dependent on the thickness of the films, as indicated in Table 1 (p < 0.05).Elastic modulus represents the stiffness of a film 28 .The results showed that films with pectin had lower elastic modulus compared to other films (66.71 ± 6.71 MPa for pectin films and 42.93 ± 11.29 MPa for pectingelatin film) which means pectin films were more flexible 29 .While films based on HPMC had the highest values (107.00 ± 15.79 MPa for HPMC-based films and 267.51 ± 41.53 MPa and 106.12 ± 39.82 MPa, for pectin-HPMC and HPMC-gelatin films, respectively).Asfaw et al. tested the mechanical properties of pectin films (3-5% w/v).They used glycerol as plasticizer in the range of 15-25%.They found pectin films had elastic modulus from 59.53 to 67.67 MPa, depending on the concentration of pectin and glycerol.This can be explained by the intermolecular hydrogen bonds between glycerol and pectin 28 .As far as HPMC, similar results were reported by Mahadevaiah et al. for HPMC films with different concentrations of glycerol and polyethylene glycol as plasticizers, with elastic modulus ranging from 1654.75 to 20.46 MPa 18 .
According to Table 1, the films which were based on one biopolymer had higher tensile strength and elongation at break compared to composite films.The chemical reactions between the polymer chains of different polymers influence the mechanical properties of the produced films 28 .Pure pectin films had higher tensile strength in comparison with composite films based on pectin-HPMC and pectin-gelatin but without statically differences (5.59 ± 1.50, 4.65 ± 0.83 and 3.19 ± 0.73 MPa, respectively).Films based on HPMC had higher tensile strength compared to all films.Tedesco et al. produced films based on HPMC, CMC and starch and reported mechanical properties, where HPMC films showed higher values 16 .According to the literature, composite films with fillers with micron size compounds have lower values on mechanical properties compared to films which are produced with smaller size fillers as nano-size compounds.This may be attributed to nano-size fillers which fill the empty areas in the matrix and build stronger interfacial bonding with the matrix 30 .In the present study, all used materials had similar size and thus no significant differences were observed between the single compound and composite films.Films based on gelatin and using glycerol as a plasticizer generally have lower tensile strength but higher values of elongation at break (3.93 ± 0.91 MPa and 17.91 ± 0.2.81%, respectively).By adding pectin in gelatin films, the tensile strength increased but without statistical difference.
The value of elongation at break represents the film's flexibility, which depends on the polymer network 27 .The composite films had lower elongation at break compared to respective one-material films (Table 1).According to Mirzaei-Mohkan et al., the addition of different compounds in the matrix of the film can change the uniformity of the structure and lead to a decrease in the stress volume tolerance in the film 31 .

Water vapor transmission rate and water vapor permeability
The calculation of the water barrier of food packaging materials is important for maintaining the quality and shelf-life of the products.Each food product requires a specific value of water content but in several cases, a reduced water transfer between the intern of the packaging and the environment is acceptable for the preservation of microbial contamination due to moisture exchange 19 .The water vapor transmission rate (WVTR) and water vapor permeability (WVP) of edible films is reported in Table 2. Packaging materials based on HPMC show high permeability of water and this is associated with its long hydrophilic chains.Hydroxyl groups can create hydrogen bonding with water molecules 23 .However, in this study, there are no significant differences in the WVTR between the films based on pectin, HPMC, and gelatin.
As far as WVP, materials based on one biopolymer showed lower values of water vapor permeability compared to composite films.Specifically, gelatin films showed the lowest value (102.00 ± 14.56 g × mm/kPa × day × m 2 ).Aitboulahsen et al. produced films from gelatin and pectin at concentration of 4% w/v when used alone and at 2% for each biopolymer when used in combination.Glycerol was added at 30% based on the concentration of biopolymer.The water vapor permeability was determined using the same methodology as the present study, but the relative humidity of the desiccator was 75%, instead of 90%.The results showed that the WVP of gelatin films and pectin films was 6.35 (± 0.01) and 6.18 (± 0.10) × 10 7 g × m −1 × Pa −1 × s −1 , respectively.When the film-forming solutions of gelatin and pectin were mixed to produce composite films the WVP reduced and was equal to 4.55 Table 1.Thickness, tensile strength (TS), elastic modulus (EM), and elongation at break (EB) of onecompound and composite films based on pectin, HPMC, and gelatin.a-e Different superscripts in the same column indicate statistically significant differences.(± 0.23) × 10 7 g × m −1 × Pa −1 × s −132 .The same result was observed in the present study, as presented in Table 2, as WVP was higher for composite materials compared to single-polymer films.In another study, the WVP of apple high methoxyl pectin films tested in 38 °C and 90% RH was 1.5(± 0.15) × 10 -9 g × m −1 × Pa −1 × s −1 , which is comparable with the present study (1.45 (± 0.12) × 10 -9 g × m −1 × Pa −1 × s −1 ) 33 .The WVP is also increased by choosing glycerol as plasticizer.The amount and type of plasticizer can also influence the water barrier as it is used to lessen the network density.Glycerol is a hydroscopic plasticizer and therefore the water content of films is increasing as the water permeation is increasing because of the high mobility of molecules 34 .

Moisture content (MC)
The moisture content of foods and their packaging is an important parameter as it is associated with the growth rate of microorganisms, keep the freshness of food and the desired taste and texture 35 .The moisture content of films based on pure pectin or gelatin was lower, compared to the composite materials.The moisture content of pure pectin film was 13.38% which was increased to 20.04% and 18.04% for blending with HPMC and gelatin film-forming solutions, respectively.Khodaei et al., reported higher values of moisture content of low methyl pectin due to the hydrophilic nature of pectin and the ability of pectin's molecules to form hydrogen bonds with water molecules.However, the moisture content of gelatin was similar with the results of the present study (11.67%) 40 .Liu et al. prepared films from gelatin using 50 mL of film forming solution at concentration of 4% (w/v) and measured the moisture content of control gelatin films equal to 14% 36 .Pure HPMC films had the higher moisture content equal to 22.13%, indicating the hydrophilicity of HPMC.Similar results were observed by Malik et al. (2022) who studied the effect of three different plasticizers on HPMC films.The moisture content of HPMC films plasticized with glycerol was 19.92% 37 .

Water solubility (WS)
The results of water solubility are presented in Table 3. Water solubility is connected with biodegradability of the films and indicates the water resistance of the films.For food that require high water resistance and have high moisture content, films with high solubility in water are not suitable packaging materials 19 .In the present study, films based on one biopolymer have higher water solubility compared to composite films, but only in gelatin films difference was statistically significant.HPMC and pectin films exhibited the highest water solubility (59.89 ± 4.83% and 60.28 ± 5.63%, respectively).HPMC films showed high water solubility, which can be attributed to hydrophilic hydroxyl groups in the HPMC films structure 38 .Cabello et al. produced pectin films with different plasticizers and determined the water solubility for different types of films.They reported that the water solubility of pectin was 9.28 ± 0.56% but when glycerol was used as plasticizer at different concentrations (0.3%, 0.5%, 1%, 3% and 5%) the water solubility of the films increased (18.35 ± 0.14%, 23.33 ± 1.27%, 49.97 ± 2.72%, 67.67 ± 1.98%, and 100%, respectively) after 12 h of immersion 29 .The water solubility of pectin films may be attributed to hydroxyl and the non-esterified carboxyl functional group of the pectin structure, which are able to build hydrogen bonds with water 39 .Gelatin films were solubilized at 44.09 ± 2.03% in water.This value was increased when HPMC and pectin were added in the gelatin-based film forming solution.Similar results were reported in the literature, i.e. 42.00 ± 0.56% by Alexandre et al. 40

Contact angle
For packaging materials, their ability to resist water absorption is a significant property.This property is indicated by water contact angle onto at the surface of the materials 43 .Additionally, the measurement of water contact angle provides information regarding the physicochemical interaction at the hydrogel interface 44 .Surfaces which have water contact angle lower than 90° (θ < 90°) are characterized as hydrophilic and they are partial or complete wetting, while surfaces which have water contact angle higher than 90° (θ > 90°) are characterized as hydrophobic 45 .
Overall, in the present study, the film which was synthesized using HPMC, was hydrophilic (θ = 56.49± 1.54°), as it is shown in Fig. 2, which is in agreement with the results reported by Tedesco et al. for HPMC based films at concentration of 7.5% with sorbitol as a plasticizer (42.42 ± 3.65°) 16 .Zhang et al. produced HPMC films at 20% concentration in distilled water, carrageenan and polyethylene glycol as solvents and reported contact angle 32.07 ± 08°, which indicates that these films were more hydrophilic 46 .HPMC contains polar functional groups of oxygen and for this reason the molecular interactions are more evident, resulting in low contact angle is low 47 .Pectin and gelatin films were hydrophobic as the contact angle in that case were 107.27 ± 2.71° and 112.15 ± 2.98°, respectively, according to Table 4.The contact angle of composite films which had HPMC as one of the materials, was lower.Pectin-HPMC films showed the lowest value equal to 18.10 ± 1.03°, indicating the presence of hydrophilic hydroxyl groups.Composite films made of gelatin as one of the materials had lower contact angle compared to pure gelatin films, but they still characterized as hydrophobic.The hydrophobic character of gelatin films can be associated with the orientation of functional groups of amino acids, during the procedure of gelification.Serine, threonine, asparagine, glutamine, aspartic acid and glutamic acid are the hydrophilic amino acids on the structure of gelatin and their functional groups are placed in aqueous positions in the film-forming solution.
Leucine, valine, phenylalanine, isoleucine and methionine are the hydrophobic amino acids on the structure of gelatin, and they are reoriented to the air-side interface, in order to form a hydrophobic surface 20 .A previous study was conducted by Ahammed et al. reporting the hydrophobic surface of gelatin films (θ = 10.48 ± 1.8°) 42 .

Color parameters
The appearance and the characteristic colors of the films which are used as food packaging materials are important parameters for consumer acceptability.The appearance of films is shown in Fig. 3.In the present study, the color of the films was investigated using instrumental (CIE L*, a* and b*) color evaluation.In Table 5, color parameters (L*, a*, b*) and total color variation (ΔΕ*) are shown.No significant differences were observed in the lightness (L*) for all types of films and the value ranged from 98.38 to 99. 25.
The a* parameter shows the green-red shade of the film.The a* value was negative for all films which indicates the green shade of the films.Blueness-yellowness is expressed by b* parameter.The yellow shade is indicated by a positive b* value.In the present study, all films, apart from HPMC film, had positive b* value.For all produced films, the ΔΕ* values were lower than 3 and the color change was not visible to the naked eye.In general, a ΔΕ* value lower than 3 indicated the transparency of the films 48 .
Compared with pure HPMC film, a significant decrease in negative values of a* and positive values of b* and ΔΕ* was observed in HPMC-gelatin and pectin gelatin films, indicating the stronger green and yellow color of the composite films.Films with one or two biopolymers based on pectin had higher values of b* parameters indicating the yellow shade of pectin as raw material.For the same reason, films based on pectin had higher ΔΕ* value, which means higher differences from the white standard.

UV-Vis barrier and light transmittance
The light barrier is an important parameter for the materials used in food packaging, as it expresses the barrier ability against ultraviolet-visible (UV-Vis) light.The wavelength range for the UV region is between 100 and 400 nm and split in three categories: UVA (315-400 nm), UVB (280-315 nm), and UVC (100-200 nm).The  www.nature.com/scientificreports/wavelength range for visible light is between 380 and 700 nm.Packaging films with good light barrier could extend the shelf-life of sensitive food products, where lipid oxidation and loss of vitamins should be prevented 32 .Table 6 presents UV-Vis light transmittance at wavelengths between 200 and 800 nm for all films.The value of transmittance at 200 nm was lower than 0.1% for all films, which indicates great barrier properties at UVC.The transmittance in the visible wavelength range (400-800 nm) was high for the films, which means that the films were transparent.Specifically, for pectin films and gelatin films, the transmittance was higher than 70%, which means that all the films were clear and transparent.This is in agreement with the study reported by Esteghal et al., where gelatin and HPMC-based films were highly transparent since they exhibited low absorbance 10 .

Microbiological evaluation of packed gilthead seabream fillets
Figure 4 represents the visual appearance of gilthead seabream fillets packed with the developed packaging films, at the day 0 and after 16 days of storage at 2 °C.The initial load of Pseudomonas spp.and Total Viable Count (TVC) was 4.10 ± 0.32 and 3.97 ± 0.33 logCFU/g, respectively.Figures 5 and 6 represent the growth curves of Pseudomonas spp.and TVC fitted to Baranyi Growth Model.For all tested films, no lag phase was observed.No significant differences were observed in microbial growth rates between the different tested packaging films (Table 7).The final microbial populations were 9.6-9.7 for Pseudomonas spp.and 9.7-10.7 for TVC.Considering the limit of acceptability of 10 7 CFU/g for TVC, the shelf-life of gilthead seabream fillets at 2°C was calculated 49 .Based on this limit, the shelf-life of gilthead seabream fillets was determined as 7-8 days at 2 °C in all tested packaging conditions, which is in agreement with the commercial use-by-date set for this product, while also with other studies in the literature evaluation the shelf-life of Mediterranean fish fillets at 0-2 °C49-51 .
The results showed that the edible packaging materials developed by pectin-gelatin, HPMC-pectin, and HPMC-gelatin may adequately reduce the use of petroleum-based polymers, such as polyvinyl chloride, and maintain a high quality of gilthead seabream fillets without affecting fish microbial spoilage and thus the shelf-life.According to the literature, gelatin-based films have been used in order to preserve animal-based products.Roy et al. produced gelatin/ager based films enhanced with antibacterial and antioxidant compounds used to pack meat over 8 days at 10 °C.The results showed that pork belly meat packed with alternative material had slower growth of total aerobic bacterial count 52 .Alirezalu et al. synthesized active chitosan films (1%) and the films used to test the effects of packed beef fillets at 4 °C for 12 days.Packed meat with chitosan films had lower TVC indicating better preservation of packed meat compared to LDPE films (7.38 ± 0.16 and 8.83 ± 0.09 logCFU/g at 12 days, respectively) 53 .Another research (Gulzar et al.) enhanced PLA films with gelatin/chitosan solutions incorporated with various concentrations of nisin.The produced PLA films coated with gelatin/chitosan and 0.4% nisin used to pack Asian seabass sliced to determine the microbial and chemical changes of stored fish at 4 °C for 12 days.The initial load of TVC of the fish packed with LDPE films and PLA-gelatin/chitosan/nisin ranged from 4.23 to 4.30 logCFU/g.In 3 days, the TVC for packed fish with LDPE was higher (6.46 logCFU/g) compared to packed fish with PLA-gelatin/chitosan/nisin (5.31 logCFU/g), indicating that the composite films preserved fish more adequately than single LDPE during refrigerated storage 54 .
The aim of the study was to replace the use of petroleum based materials without reducing the shelf-life of the packed fish.Pectin, HPMC and gelatin films do not exhibit antimicrobial property activity, if no antimicrobial compounds have been incorporated in the matrix of biopolymers, but they can help to release a wide range of active compounds 55 .Other studies investigated the antimicrobial activity of the films when combined with antimicrobial compounds.Shivangi et al. studied the antimicrobial activity of pectin films against Pseudomonas aeruginosa and Bacillus cereus, and they pointed antimicrobial activity against the two microorganisms only when crude mulberry leaf extract, deoxynojirimycin and chlorogenic acid were added.In the case of pure pectin films without extracts no activity against the microorganisms was observed 24 .According to Möller et al., HPMC exhibits antimicrobial activity when it is combined with chitosan 56 .Leite et al., tested the antimicrobial activity of gelatin films and they pointed that the films without nonoxidized tannic acid (nTA) had not inhibitory effect against Staphylococcus aureus and Escherichia coli growth 57 .
Future studies should be conducted focusing on the incorporation of antimicrobial compounds such as essential oils, Zn ions, or silver nanoparticles (AgNPs), with the aim to address the effects of using active packaging materials on the shelf-life of gilthead seabream fillets.Table 6.UV-Vis light transmittance (200-800 nm) of films based on pectin, HPMC, gelatin and pectin-HPMC, pectin-gelatin, and HPMC-gelatin.a-f Different superscripts in the same column indicate statistically significant differences.

Conclusions
Alternative edible and biodegradable food packaging films were developed using pectin, gelatin, and HPMC which could replace petroleum-based packaging materials, such as PVC.All the tested films showed excellent barrier properties in the UVC wavelengths and good performance in the UVA and UVB wavelengths range.The obtained results showed the potential of synthesized films made from polysaccharides and proteins (HPMC, pectin and gelatin) as green environmentally friendly packaging materials to adequately preserve fresh gilthead seabream fillets during refrigerated storage at 2°C, in terms of microbial spoilage.

Preparation and characterization of films
The films were produced according to the solvent casting method.2% w/v of each biopolymer was dissolved into water and mixed for 30 min.Gelatin and pectin solution was stirred under 35 °C and the HPMC solution was stirred under 80 °C for 30 min.Then, glycerol was added at 30% of the weight of each biopolymer and mixed for 15 min.The HPMC solution was stirred for 1 h and the temperature was decreased to 35 °C before adding glycerol to increase the transparency of the films.The solutions were mixed in proportions of 1:1 and three additional types of film-forming solution were produced.In total, six different types of film-forming solutions were produced, three based on one biopolymer (pectin, HPMC, gelatin) and three composites (pectin-HPMC, pectin-gelatin, HPMC-gelatin).All solutions were centrifugated at 4000 rpm, 25 °C for 10 min.After centrifugation 20 g of each solution was poured into plastic petri dishes with diameter 14 mm 19 .

Figure 6 .
Figure 6.Total Viable count in seabream fillets packed with composite films based on pectin, gelatin, and HPMC when stored at 2 °C compared with seabream fillets packed with PVC films.

Table 2 .
42d 31.1% by De Carvalho and Grosso41.Ahammed et al. produced films with 8% gelatin and reported total solubility in the water42.Water vapor transmission rate ( g day ×m 2 ) and water vapor permeability (g × m −1 × Pa −1 × day −1 ).a-c Different superscripts in the same column indicate statistically significant differences.

Table 7 .
Kinetic parameters of microbial growth (growth rate, k; maximum population; N max ) for Pseudomonas spp.and TVC in gilthead seabream fillets packed with composite films based on pectin, gelatin, and HPMC.a,b Different superscripts in the same column indicate statistically significant differences.